Regulatory Mechanisms of the MicroRNA Pathway

MicroRNAs (miRNAs) associate with members of the Argonaute protein family and downregulate partially complementary messenger RNAs (mRNAs) (1). miRNA activity is tightly regulated during development and in normal physiologic settings, while gain or loss of these control mechanisms can contribute to disease (2-4). To identify new mechanisms that regulate the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening (5, 6) coupled with a fluorescent miRNA pathway reporter. These experiments revealed an unanticipated role for the ANKRD52-PPP6C serine/threonine phosphatase complex as a critical regulator of miRNA activity in human cells. Loss of this complex significantly impaired global miRNA function. Genetic and biochemical studies revealed that phosphorylation of Argonaute2 (AGO2) on a set of highly conserved serine residues, S824-S834, blocks target mRNA engagement. Constitutive activity of the ANKRD52-PPP6C complex is necessary to remove these inhibitory phosphates and thereby allow miRNA-mediated silencing. A genome-wide CRISPR-Cas9 suppressor screen performed in ANKRD52-/- cells identified CSNK1A1 as the inhibitory AGO2 kinase that phosphorylates these sites. Together, these findings reveal a previously uncharacterized AGO2 phosphorylation cycle, uncovering a major mechanism through which the miRNA pathway is regulated and highlighting the power of iterative CRISPR-Cas9 screening for the dissection of biological pathways directly in human cells.